Studies on asexual stage immunity to P. falciparum 1. Search for conserved, parasite-encoded epitopes on the surface of malaria IEs Pregnancy associated malaria (PAM) remains a major threat to women and their unborn children in endemic areas. IEs accumulate in the placenta and the ensuing inflammatory response can increase the likelihood of anemia, hypertension, premature delivery and possible death of low birth weight infants. Clinical immunity to PAM in multigravid women has been attributed to antibodies that recognize VAR2CSA, a large multi-domain variant of the PfEMP-1 family, which binds to chondroitin sulfate A (CSA), on the surface of red cells. Opsonization of infected erythrocytes by cytophilic antibodies that recognize VAR2CSA epitopes represents an understudied host effector mechanism in PAM. We have established a flow cytometry-based opsonization assay and we have shown that, in contrast to malaria-nave American adults and malaria-exposed Malian men, purified IgGs from multigravid Malian women showed higher 1) reactivity to recombinant DBL domains by ELISA, 2) binding to red cells expressing VAR2CSA, and 3) opsonization of these infected erythrocytes by human monocytic cells. Importantly, preincubation of IgGs from multigravid women with selected VAR2CSA domains significantly diminished opsonization of VAR2CSA-expressing IEs by monocytes. Antigen reversal of opsonization provides the first evidence that domains DBL3x, DBL5&#949;, and DBL2x, are the primary targets of these opsonizing antibodies. Our study focuses attention on these domains for PAM vaccine development and introduces a new tool to identify targets of host effector responses to pathogens. 2. Investigate targets of merozoite immunity a. AMA1 -While AMA1 is a prominent vaccine candidate, it has two issues:1) antigenic polymorphism so that antibody responses tend to be strain-specific and 2) insufficient antibody production to result in protective immunity in humans. Our data as well as that of our collaborators supports the use of a mixture of 4-5 different AMA1 alleles to overcome the polymorphism and elicit broadly reactive antibodies. Further, we have collaborated with LMVR investigators to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in a standardized parasite growth inhibition assay (GIA). This complex is required for triggering junction formation between the merozoite and the red cell surface. Evaluation of this combination in an immunization-challenge trial in a non-human primate (Aotus) is ongoing to obtain supportive data for a human clinical trial. b. We continue to work with other investigators on two different merozoite protein families - the reticulocyte binding-like family (RBL) and the erythrocyte binding-like (EBL) proteins as novel candidates either as single proteins or as mixtures. We have collaborated with others from LIG to show that antibodies to RH5 isolated from those living in Mali do have GIA activity, lending support to this protein as a new vaccine candidate. Also we have evaluated the GIA activity of antibodies to both RH5 and the RIPr protein (RH5 interacting protein) which appear together on the surface of the invading merozoite. Each protein elicits strong GIA activity. 3. Studies of immunity to malaria in Kenieroba, Mali. Our 4-year investigation of the acquisition of immunity to malaria in Malian children represents perhaps the most detailed longitudinal study of malaria in African children, and it reveals the impact of the sickle cell trait (HbAS) in protection against malaria. We have evaluated antibody responses of these children in several different assays including ELISA and GIA, and in 2014 we are adding other functional measures such as the neutrophil-dependent antibody-dependent respiratory burst assay and opsonization assays to obtain a broad scope of anti-parasite functional activities. Studies on parasite sexual stages and malaria transmission: 1. Develop quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity and extend this to parasites in the field. The gold standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the SMFA, and we have performed an in depth study of this assay to define its characteristics. We have shown that the SMFA is quite reproducible at high concentrations of antibody but highly variable at low concentrations and we have worked with the Biostatistics group at NIAID to develop a computer model of the assay. In 2014 we have worked with others at LMVR and at FDA to develop improved read-outs for this assay. 2. In 2014 we used the SMFA assay to assess whether children immunized with the RTS,S vaccine (the most advanced malaria vaccine candidat) could develop antibodies that would block transmission. We were not able to show an impact of the polyclonal human or monoclonal antibodies on transmission to mosquitoes whether measured by reductions in oocyst density or sporozoites in salivary glands. 3. Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA in 2014 we have evaluated a number of potential transmission blocking vaccine candidates as well as different presentations of these antigens. In collaboration with colleagues at Oxford University we have produced and tested a number of known and unknown sexual stage proteins from P. falciparum. These proteins have been produced in HEK293 cells and the products were used to immunize mice. Sera from these animals are being evaluated in the SMFA. 4. Assess the presence of asexual and sexual stage parasites in Kenieroba residents throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) of malaria transmission in Kenieroba to address the limited information on actual transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study. Volunteers were finger pricked twice per month for 1 year to collect DNA and RNA. During this year we have worked out the methodology for the PCR to detect parasite DNA on filter papers and also the RT-PCR procedure to identify RNA specific for gametocytes. Using these techniques, we are evaluating the carriage of parasite DNA as well as gametocytes in 500 villagers of all ages. This will identify those individuals capable of malaria transmission who might be targeted for intervention. 5. Determine whether Kenieroba, Mali residents develop antibodies which have blocking activity in SMFA. The Kenieroba study of transmission includes a venipuncture sample from all the volunteers 3 times during the year. These larger volume serum samples will be used in SMFA to determine whether antibodies have transmission blocking activity. 6. Determine the relationship between the SMFA assay and the DMFA, which measures the transmission blocking activity of antibodies to sexual stage parasites from infected people in the field. We are evaluating whether models that we have developed that work with SMFA are also applicable to DMFA. Other studies: 1) We have collaborated with Dr. Akhil Vaidya of Drexel University in showing that asexual P. falciparum parasites do not require synthesis of heme, but it is necessary for sexual stage differentiation and infection of mosquitoes; 2) In collaboration with Dr. Olivier Lichtarge at Baylor University we have provided supporting data for a new strategy to identify function of unknown genes(supergenomic network compression) and have aided evaluation of PfEXP1 as a glutathione transferase which can be inhibited by artesunate;3)We continue to work with Drs. Amy Bei and Dyann Wirth to profile the changes in P. falciparum clonal frequency in Senegal and the influence of immune responses on that process.